Trypanosomatid parasites of the genus Leishmania infect close to 15 million people world-wide. Diagnosis is difficult in endemic areas because of the currently available detection schemes are inadequate because of lack of sensitivity, and time intensiveness. Recent concern regarding possible link between the Gulfwar syndrome and Leishmaniasis has accentuated the lack of and need for better diagnostic modalities for both patients and blood products. A more reliable target for detection is the parasite's own mitochondrial DNA (kDNA) which remains constant through its life cycle and is present in thousands of copies per cell. We have characterized kDNA sequences from eight different Leishmania isolates and have used a PCR-based scheme to design detection systems for several species. This techinque can readily detect Leishmania kDNA from single cell in the presence of vast amounts of human DNA. The utility of these PCR schemes has been further shown by their ability to positively identify parasite isolates from kala-azar patients and individuals with cutaneous infections. Based on our experience with the PCR-based detection schemes, we have developed a PCR assay that is capable of amplifying kDNA of L. donovani in a species-specific manner among Old World Leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of Kala-azar (KA) patients and from skin lesions of Post kala-azar dermal leishmaniasis (PKDL) patients. A total of 107 clinical samples of Leishmaniasis were analyzed. Of these 102 (95.3%) were positive in PCR. The test provided for diagnosis of KA with 96% sensitivity using patient whole blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45/48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the endemic area viz., M. tuberculosis, M. leprae and Plasmodium could be ruled out. Eighty one control samples including dermal scrapings from normal portions of skin from PKDL patients were all negative. 2/20 endemic controls were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus this PCR assay represents a tool for the diagnosis of KA and PKDL in patients in a non-invasive manner, with simultaneous species identification of parasite in clinical samples.